Exploring RNA structures by means of molecular dynamics simulations: the flavin mononucleotide aptamer, coaxial stacking in a nicked hairpin and water-mediated base pairs in a duplex

Es wurden Molekulardynamik Simulationen von verschiedenen RNA Strukturen durchgefuehrt, u.a. von einem FMN-RNA Aptamer, ein Modell zum Koaxialen Stacking und eine Helix mit 2 wasservermittelten Basenpaaren

Role of Transcription in Mammalian Copy Number Variant Formation

Genome instability, defined as an increased tendency of genome alteration, is the cause of many human diseases and conditions. It is a hallmark of human cancer and plays a role in aging and the development and function of the nervous system. Genome instability can manifest in several ways, including gaps and breaks at Common Fragile Sites (CFSs) and Copy Number Variants (CNVs). CFSs are sites on human metaphase chromosomes prone to forming gaps or breaks following replication stress. CNVs are submicroscopic genomic alternations that change the copy number of the affected region, also often following replication stress. The genome regions most prone to replication stress-induced CNVs, called “hotspots,” coincide with CFSs. In spite of their implications for human health, mechanisms leading to instability at CFSs and CNV hotspots are unclear. CFSs/CNV hotspots are AT-rich and late replicating, but those properties are not sufficient for the sites’ instability. DNA sequence at CFSs/CNV hotspots is shared among all cells, but instability is cell line-specific. We also found that while about 20% of the genome replicates late, hotspots only comprise 0.4% of the genome. Hence, instability at hotspots is determined by properties that vary between different cell lines and genomic regions. Transcription is one such property. We found that CFSs/CNV hotspots are enriched in large (>500kb), transcribed genes and that given a cell line’s transcription profile we can predict where CFSs/CNV hotspots will be in that cell line. I further show that abrogating expression of a large hotspot gene leads to a reduced number of aphidicolin-induced CNVs. These results established transcription of large genes as a determining factor for instability at hotspots. We propose that a conflict between transcription of large genes and DNA replication drives hotspot instability. I tested a model in which R-loops (RNA/DNA hybrids) create a physical interference for the replication fork and cause the fork to stall and initiate genomic alteration. R-loop manipulation by altering expression of RNase H1 had no significant effect on the frequency of APH-induced instability at hotspots, implying that R-loops do not play a central role in driving APH-induced CNVs, unlike a prior study showing that R-loop manipulation changes CFS instability. However, R-loop accumulation changes the location of breakpoints of these CNVs and change the frequency of the spontaneous CNVs, suggesting that R-loops may still play a role in both APH-induced and spontaneous CNV formation. In sum, the studies in this dissertation reveal that transcription of unusually large genes plays a pivotal role in instability at CFSs/CNV hotspots during replication stress, but not via an R-loop-associated mechanism. Nonetheless, R-loops threaten genome instability and affect CNV formation outside of hotspots. Future studies are necessary to explore other transcription-replication conflict models at CFSs/CNV hotspots and further characterize R-loop induced CNVs.PHDHuman GeneticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147729/1/sohae_1.pd

The orientation and dynamics of the C2'-OH and hydration of RNA and DNA.RNA hybrids.

The stereochemical and dynamic properties of the C2' hydroxyl group in several DNA.RNA hybrids have been measured by NMR and compared with the homologous RNA duplex. The C2'-OH NMR signals of the RNA strands were identified, and numerous specific assignments were made. The rate constants for exchange of the hydroxyl protons with water were determined at 5 degrees C, and were found to depend on both the position within a particular sequence and the nature of the duplex. On average, the exchange rate constants were slowest for the hybrids of composition rR.dY, and fastest for the RNA duplex, with an overall range of approximately 10-50/s. In the DNA.RNA hybrids, strong NOEs and ROEs were observed between the OH and the H1' of the same sugar, unambiguously showing that the OH proton points toward the H1' most of the time, and not toward the O3' of the same sugar. Evidence for significant hydration in both grooves of the DNA.RNA hybrids and the DNA duplex was found in ROESY and NOESY experiments. On average, the minor groove of the DNA.RNA hybrids showed more kinetically significant hydration than the DNA, which can be attributed to the hydrophilic lining of hydroxyl groups in RNA

Developing metagenomic methods for legionella detection

Legionella is a Gram-negative bacterium naturally present in freshwater and soil. The bacteria can enter, colonise and multiply in man-made water systems. Infection through inhalation of aerosols containing the bacteria can cause Legionnaires' disease (LD) an atypical, severe pneumonia in individuals with underlying risk factors.Legionella pneumophila serogroup 1, the most widely studied species, is reported to account for more than 90 % of all clinical isolates related to LD in England. L. pneumophila is isolated routinely at Public Health England however Legionella is a slow growing bacterium, typically taking from three to five days to grow. Additionally, it has been reported that L. pneumophila is isolated in only 60 % of urinary antigen-positive cases. Here, the application of metagenomic sequencing was investigated in Legionella positive clinical and environmental samples, the hypothesis being that metagenomic sequencing may provide a more time efficient result and may reveal previously undetected heterogeneity in clinical and environmental cases. The results demonstrate that L. pneumophila genomes can be captured and sequenced from patients with LD and from environmental source samples without prior culture using a targeted capture approach. The data generated also demonstrate that Legionella diversity within environmental sources as well as a clinical case could be captured. Importantly, the work has demonstrated the first successful application of in silico 7-loci sequence-based typing and 50 core gene MLST to Legionella data generated by a metagenomic method. Overall, this thesis demonstrates the proof of concept of targeted metagenomic sequencing of L. pneumophila directly from multiple patients and environmental sources as well as the ability to capture a variety of sequence types. Furthermore, the challenges of implementing metagenomic sequencing for routine diagnostic use and future avenues for technical optimisation of the targeted capture approach are outlined.Open Acces

Ribonuclease H2, RNA:DNA hybrids and innate immunity

The activation of the innate immune system is the first line of host defence against infection. Nucleic acids can potently stimulate this response and trigger a series of signalling cascades leading to cytokine production and the establishment of an inflammatory state. Mutations in genes encoding nucleases have been identified in patients with autoimmune diseases, including Aicardi-Goutières syndrome (AGS). This rare childhood inflammatory disorder is characterised by the presence of high levels of the antiviral cytokine interferon-α in the cerebrospinal fluid and blood, which is thought to be produced as a consequence of the activation of the innate immunity by unprocessed self-nucleic acids. This thesis therefore aimed to define the role of one of the AGS nucleases, the Ribonuclease H2 (RNase H2) complex, in innate immunity, and to establish if nucleic acid substrates of this enzyme were able to induce type I interferon production in vitro. The AGS nucleases may function as components of the innate immune response to nucleic acids. Consistent with this hypothesis, RNase H2 was constitutively expressed in immune cells, however, its expression was not upregulated in response to type I interferons. RNase H2-deficient cells responded normally to a range of nucleic acid PAMPs, which implied that a role for RNase H2 as a negative regulator of the immune response was unlikely, in contrast to the reported cellular functions of two other AGS proteins, TREX1 and SAMHD1. Therefore, no clear evidence was found for the direct involvement of RNase H2 in the innate immune response to nucleic acids. An alternative model for the pathogenesis of disease hypothesises that decreased RNase H2 activity within the cell results in an accumulation of RNA:DNA hybrids. To investigate the immunostimulatory potential of such substrates, RNA:DNA hybrids with different physiochemical properties were designed and synthesised. Methods to purify the hybrids from other contaminating nucleic acid species were established and their capacity as activators of the innate immune response tested using a range of in vitro cellular systems. A GU-rich 60 bp RNA:DNA hybrid was shown to be an effective activator of a pro-inflammatory cytokine response exclusively in Flt3-L bone marrow cultures. This response was completely dependent on signalling involving MyD88 and/or Trif, however the specific receptor involved remains to be determined. Reduced cellular RNase H2 activity did not affect the ability of Flt3-L cultures to mount a cytokine response against the RNA:DNA hybrid. These in vitro studies suggested that RNA:DNA hybrids may be a novel nucleic acid PAMP. Taken together, the data in this thesis suggest that the cellular function of RNase H2 is in the suppression of substrate formation rather than as a component of the immune response pathways. Future studies to identify endogenous immunostimulatory RNA:DNA hybrids and the signalling pathways activated by them should provide a detailed understanding of the molecular mechanisms involved in the pathogenesis of AGS and related autoimmune diseases